Lyme disease is the most common vector born infectious disease in North America, Asia and Europe. It is a multisystem, inflammatory, progressive disease with a wide range of clinical manifestations, sometimes including erythema migrans (“EM”) the initial and readily recognized cutaneous “target” lesion. From EM, which may only present in an estimated 50% of the patients, it may disseminate to other organs, including the nervous system, joints, and heart (http://www.ilads.org/lyme/lyme-quickfacts.php). Such progression may result in permanent neurological and/or musculoskeletal damage, and debilitating symptoms including fatigue and other flu-like symptoms.
A diagnosis of LD is currently based on clinical symptoms and serology, i.e., antibody to LD. The latter may not be detectable during the first few weeks of infection. The current LD diagnosis methodology uses a recommended two-tier serological assay, misses up to 60% of early infections, and is unable to distinguish a past infection from current, active Borrelia infection (Hinkley et al, (2014) Clinical Infectious Diseases 59(5):676). More specifically, current sero-diagnostic assays include an ELISA assay to detect antibodies to Borrelia species followed by a Western blot for confirmation. If diagnosed in the early stages, the disease can generally be cured with therapeutic agents, e.g., antibiotics. If left untreated, complications involving joints, the heart, and the nervous system can occur. It is therefore crucial to be able to specifically detect and differentiate Lyme disease from other diseases in order to effectively treat it and avoid complications that may develop in later stages. The instant invention includes a method that employs two or more biomatkers to differentiate, diagnose and treat LD.
Due to the fact that few spirochetes are present, i.e., Borrelia burgdorferi, the spirochete that causes LD, especially in blood specimens, the best current methods measure immune response. While not fully reviewed here, many steps, including antigen capture by “professional antigen presenting cells” and numerous signaling and processing steps, as well as coordination of various immune cell types, are required prior to production of Ab. For example, prior to Ab secretion numerous immune signaling agents must be secreted and transported from one type of immune cell to another. An immune network including APCs, T-cells of various types and B-cells is generated. Even once these signals are generated numerous steps of B-cell maturation, transcription, translation, processing and secretion are required before even low levels of IgM Ab are present in the blood. Evolution has driven the spirochete to attempt to evade and suppress the Ab response, and numerous steps between the APC and the Ab provide many possible opportunities for suppression. Certain subjects, i.e., genotypes, may also be less effective in promptly completing the process and producing high levels of Ab. Thus, measurement of the earliest steps of immune response, prior to antibody excess that can be measured in the blood, is rationally expected to a more sensitive method of differentiating LD from other diseases.
Although the Ab response may be delayed or weak, a significant number of acute Lyme disease patients have such a florid early immune response that it may be visible with the naked eye as erythema migrans (EM). It is known that some clinicians may miss EM, and some skin types may not show the response. Further, it should be noted that EM detected clinically may not be indicative of Lyme disease. For examples of descriptions and differential diagnosis based on apparent EM, see, e.g., Hsu (2001) Am Fain Physician 64(2):289; and, Nopper (1998) Pediatr Ann 27:136. Other symptoms of initial Lyme disease including, fever, malaise, arthralgia, headache and stiff neck are even less specific than EM.
Currently, there is no accurate way to differentiate other than process of elimination or differential diagnosis methodologies, i.e., epidemiology based or likelihood-ratio based methods.
The signs and symptoms of Lyme disease, other than EM, are non-specific, they may include fever, malaise, body aches, joint pain, neck pain, headache and sore throat. Since EM is present in only the minority of the cases, the clinical diagnosis is very unreliable. While there are specific diagnostic tests for some of the other diseases that cause similar symptoms it is often difficult or impossible to determine what is causing the problem. For example tests for strep throat may be done by a physician on site but they are unreliable. Laboratory tests for other viruses or bacterial infections are possible, however a number of tests would need to be ordered and the results are of variable quality. Further not all the possible items in a differential diagnosis can be easily tested for. As an example cancer is in the differential diagnosis as a common cause of fatigue and it is very difficult to test for all possible causes of cancer; even if a large number of very expensive and time consuming scans are done early cancers are still often missed. Most importantly Lyme disease would still remain a diagnosis of exclusion. In order to definitively separate Lyme disease from other diseases, accurately, in a reasonable period of time and with high specificity a specific diagnostic test is needed.
The significant disadvantages (poor sensitivity and specificity) of current assays lead to a significant medical need for better diagnostic tests for differentiating, diagnosing, and treating Lyme disease. There are some methods that culture blood cells and look for cytokine production in response to antigens for the detection of LD, however these are expensive, labor intensive, rely on artful methods, and require living white blood cells. Molecular proteomic methods of identifying the immune mediators of an early immune response, even when the presenting symptoms are substantially similar to a number of other diseases and/or infections, are targeted in the present invention. These molecular methods are more sensitive and accurate than clinical observation for the purpose of differential diagnosis and precede the presence of positive serology.
A differential diagnosis is typically defined as diagnostic procedures used for distinguishing a disease or condition from others that present similar symptoms. Such procedures are used to diagnose a specific disease and/or eliminate conditions. More specifically, differential diagnostic procedures are systematic diagnostic methods used to identify the presence of a disease where multiple alternatives are possible, wherein a process of elimination is employed that reduces the probability of possible conditions. Historically, evidence such as symptoms, patient history, and medical knowledge and experience are utilized. To date there is not an accurate and effective molecular format for differential diagnosis of LD.
LD is difficult to diagnose because there are myriad conditions and syndromes that have overlapping symptoms, for example, Amyotrophic Lateral Sclerosis, Chronic Fatigue Syndrome, Fibromyalgia, Leptospirosis, Multiple Sclerosis, Rheumatoid. Arthritis, viral infection including flu and colds, bacterial infection, spider bites, skin lesions, etc. Some of these, such as Chronic Fatigue Syndrome, are themselves diagnoses of exclusion for which no tests are available. While many of these conditions might be ruled out via the inefficient diagnostic process of elimination, there remains a need for differentiation and/or diagnosis and/or treatment of LD via molecular methods, and specifically via biomarker signatures capable of differentiating LD from other diseases. In addition to differentiating LD from other diseases, identification of an immune biomarker signature that differentiates LD may also aid in identification of key immunologic pathways that may be targeted for therapeutic purposes. More specifically, biomarker analysis can provide prognostic as well as diagnostic information, guide initial treatment choice, monitor treatment efficacy, and improve outcomes. The two or more biomarkers, hereinafter referred to as the biomarker signature, of the instant invention allow specifically for differentiation from other diseases, disease diagnosis, effective treatment, and progression prevention. Differentiation, diagnosis, appropriate treatment, and prevention of progression and/or recurrence can play a role in all of these disease management areas. The poor sensitivity and specificity of the current methods for detecting LD means that methods for the diagnosing, prognosing, monitoring, differentiating, treating, and managing of Lyme disease in a subject characterized by the detection of a biomarker signature comprised of a combination of two or more analytes differentiating LD from other diseases presenting similar symptoms would be an invaluable tool to aid clinicians. Such methods have the potential to expedite and increase the accuracy of LD diagnosis and treatment.
A method such as discussed herein, that uses, for example, serum, plasma, blood, blood spots, blood filtrate, urine, saliva or tears, to detect the in vivo production of a biomarker signature, i.e., cytokine or other analyte markers, would be easier, more generally applicable, and more accurate than those currently available. Both highly specific and more sensitive tests detecting biomarker signatures may also have value as a component of a multi-tier LD assay. Such a biomarker assay would be more sensitive and more specific than current serological assays, and would provide detection, differentiation, and diagnosis at early time points, i.e., earlier than detection of antibodies. The present invention is an effective diagnostic method for differentiation of LD from other diseases exhibiting similar symptoms, e.g., bacterial, viral, autoimmune, that improves disease outcomes in patients.